3-dimensional klinostat for culture of cells

ABSTRACT

In order to grow an object to be cultivated or grown, a growth object is encapsulated in a vessel, and the growth object is grown without substantial influence of gravity.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a technique using a klinostat, and moreparticularly, to a method of cultivating a cell lump to form anartificial organ and a method of forming material, using a klinostat.

2. Description of the Related Art

A medical technique is important to recover functions of a damagedtissue or organ using artificially cultivated tissue and organ, i.e., anartificial organ in future regeneration medicine. In order that themedical technique is realized, cell cultivation technique forcultivating a tissue or organ with a 3-dimensional structure from a cellor cell lump is indispensably necessary.

As such a cell cultivation technique, a technique is known in which acell lump is encapsulated in a cultivation vessel filled with culturefluid, and is held in a floating state by rotating the cultivationvessel around one axis, applying vibration to the cultivation vessel,introducing air bubbles into the cultivation vessel or generating awatercourse with an impeller such that the cell lump is grown in theflowing state. In this conventional technique, however, the cell lumpdisperses and the cell lump suffers damage through the contact with thecultivation vessel wall. Therefore, the cultivation of a 3-dimensionaltissue is supposed to be obstructed.

Also, a technique is studied in which a cell lump is cultivated in aminute gravity environment like the universe, as another cellcultivation technique. It is considered that it would be possible tocultivate a tissue with the 3-dimensional structure, because the celllump does not sink under the minute gravity environment. Also, thepossibility of the forming of a cell aggregate of a high density isshown in the minute gravity environment. However, it lacks of thepracticality to cultivate a cell lump in the universe.

Also, an animal and plant growing apparatus is known in JapaneseExamined Patent application (JP-B-Heisei 7-89798). In the animal andplant growing apparatus of this reference, a vessel to store an animaland plant is rotated around two or more 2 axes so that gravity isapplied to the animal and plant into many directions. The animal andplant is grown in a pseudo gravity free environment.

Also, Japanese Examined Patent application (JP-B-Heisei 7-89798)discloses to grow an animal and plant and to cultivate a cell. However,a specific method to cultivate a cell is not disclosed.

SUMMARY OF THE INVENTION

Therefore, an object of the present invention is to provide a practicaltechnique to cultivate a tissue with a 3-dimensional structure.

Another object of the present invention is to provide a technique inwhich cell dispersing can be prevented when a tissue with the3-dimensional structure is formed through cultivation of a cell.

Another object of the present invention is to provide a technique whicha cell can be cultivated for a long term when a tissue with the3-dimensional structure is formed through cultivation of the cell.

Another object of the present invention is to provide a technique whichthe environment of cultivation of a cell can be optimized when a tissuewith the 3-dimensional structure is formed through the cultivation ofthe cell.

Another object of the present invention is to provide a technique forforming a large tissue with the 3-dimensional structure is formedthrough the cultivation of the cell.

In an aspect of the present invention, in order to grow an object to becultivated or grown, a growth object is encapsulated in a vessel; andthe growth object is grown without substantial influence of gravity.

When the growth object is a cell lump, the cell lump and fluid may beencapsulated in the vessel. Also, when the growth object is a cell lumpand the cell lump is adhered to an artificial matrix, the fluid, theartificial matrix, and a supporting section for supporting theartificial matrix may be encapsulated in the vessel.

Also, the artificial matrix to which the cultivated cell lump is adheredis desirably taken out as an artificial organ, after the cultivation.

Also, differentiation of the cell lump is desirably promoted.

Also, the coefficient of viscosity of the fluid is desirably adjustedsuch that the cell lump does not touch the vessel.

Also, the vessel is rotated around different n axes (n is an integermore than 1) such that the influence of the gravity is eliminated.

Also, it is desirable that the cell lump is cultivated while supplyingthe fluid to the vessel. In this case, the fluid may be circulated ordumped.

Also, when the growth object is an organism, fluid, containing materialnecessary for growth of the organism, is supplied into the vessel. Inthis case, the vessel is desirably rotated around n axes (n is aninteger more than 1).

In another aspect of the present invention, culture fluid and a celllump are encapsulated in a cultivation vessel, and the cell lump iscultivated without influence of gravity. The cultivation cell lump isheld in a floating state in the culture fluid.

Also, the cultivation cell lump may be adhered to an artificial matrix.The culture fluid, the artificial matrix to which the cell lump isadhered, and a supporting section to support the artificial matrix isencapsulated in the cultivation vessel.

Also, the cultivation vessel is desirably rotated around different naxes (n is an integer more than 1).

The method may further include adjusting a coefficient of viscosity ofthe culture fluid such that the cell lump does not touch the cultivationvessel while the cultivation vessel is rotated around the n axes. Also,the method may further include promoting differentiation of the celllump.

The artificial matrix to which the cultivated cell lump is adhered istaken out as an artificial organ after the cultivation.

In another aspect of the present invention, culture fluid and acultivation cell lump are encapsulated in a cultivation vessel, and thecultivation vessel is rotated around n axes (n is an integer more than1). The cultivation cell lump is held in a floating state in the culturefluid.

The coefficient of viscosity of the culture fluid is desirably adjustedsuch that the cultivation cell lump does not touch the cultivationvessel while the cultivation vessel is rotated around the n axes.

The culture fluid, an artificial matrix to which cultivation cell lumpsare adhered, and a supporting section to fixedly support the artificialmatrix are encapsulated in a cultivation vessel. The cultivation vesselis rotated around n axes (n is an integer more than 1). Also,differentiation of the cultivation cell lumps is promoted.

In another aspect of the present invention, culture fluid, cultivationcell lumps are encapsulated in a cultivation vessel, and the cultivationcell lumps is cultivated in a state which the cultivation vessel isrotated around n axes (n is an integer more than 1), to form anartificial organ. The cultivation cell lump is held in a floating statein the culture fluid.

The coefficient of viscosity of the culture fluid may be desirablyadjusted such that the cultivation cell lumps do not touch thecultivation vessel while the cultivation vessel is rotated around the naxes.

In another aspect of the present invention, culture fluid, an artificialmatrix to which cultivation cell lumps are adhered, and a supportingsection to fixedly support the artificial matrix are encapsulated in acultivation vessel. The cultivation cell lumps is cultivated in arotation state of the cultivation vessel around n axes (n is an integermore than 1). The structure of the cultivated cultivation cell lumps andthe artificial matrix is taken out as an artificial organ after thecultivation.

Also, differentiation of the cultivation cell lumps is desirablypromoted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the cell cultivation apparatus according toa first embodiment of the present invention;

FIG. 2 is the outward appearance photograph of a cell lump cultivated bythe cell cultivation apparatus in the first embodiment;

FIG. 3 is a cross sectional photograph of the cell lump cultivated bythe cell cultivation apparatus in the first embodiment;

FIG. 4 is a diagram showing the cell cultivation apparatus according toa second embodiment of the present invention;

FIG. 5 is a diagram showing a rotary joint;

FIG. 6 is a diagram showing a cultivation vessel;

FIG. 7 is a diagram showing the cultivation vessel 42 used in the cellcultivation apparatus according to a third embodiment of the presentinvention;

FIG. 8 is a diagram showing a modification of the cultivation vessel;

FIG. 9 is a diagram showing the structure of a growth vessel 51 used inthe third embodiment of the present invention;

FIG. 10 is a diagram showing the structure of a growth vessel 54 used inthe fourth embodiment of the present invention;

FIG. 11 is a diagram showing the structure of a modification of a growthvessel; and

FIG. 12 is a diagram showing the structure of a growth vessel 71 used inthe fifth embodiment of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, a method of cultivating a cell according to the presentinvention will be described with reference to the attached drawings. Thepresent invention relates to the U.S. patent application Ser. No.(10/119,895) filed Apr. 11, 2002. The disclosure of the application isincorporated herein by reference. Also, the present invention relates tothe US patent application Ser. No. 10/233,566 entitled “APPLICATIONAPPARATUS OF 3-DIMENSIONAL KLINOSTAT AND GROWING METHOD USING THE SAME”and claiming the priority based on Japanese patent application2001-267539. The disclosure of the application is incorporated herein byreference.

(First Embodiment)

In the method of cultivating the cell according to the first embodimentof the present invention, the cell cultivation apparatus 10 shown inFIG. 1 is used. The cell cultivation apparatus 10 is comprised of a3-dimensional klinostat 1 and a cultivation vessel 2. The 3-dimensionalklinostat 1 rotates the cultivation vessel 2 around n axes (n is aninteger more than 1) (n=2 in this embodiment). Culture fluid and acultivation cell lump are encapsulated in the cultivation vessel 2. Thecultivation cell is not fixed on the cultivation vessel 2 and floats inthe culture fluid. A differentiation factor like activin is added to theculture fluid to promote the differentiation to the desired tissue.

The 3-dimensional klinostat 1 is composed of a main unit 3, a motor 4,an outer frame 5, a motor 6 and an inner frame 7. The main unit 3 isinstalled in a stationary system. The main unit 3 is composed of a base3 a and legs 3 b and 3 c. The legs 3 b and 3 c are connected with thebase 3 a. The motor 4 is provided for the leg 3 b. The motor 4 isconnected with the outer frame 5 to rotate the outer frame 5 around arotation axis 4 a. The motor 6 is provided for the outer frame 5. Themotor 6 is connected with the inner frame 7 to rotate the inner frame 7around a rotation axis 6 a. The rotation axis 6 a is substantiallyorthogonal to the rotation axis 4 a. The above-mentioned cultivationvessel 2 is connected with the inner frame 7. The cultivation vessel 2is in the neighborhood of the intersection of the rotation axis 4 a andthe rotation axis 6 a. The cultivation vessel 2 is rotated together withthe inner frame 7. When the outer frame 5 and the inner frame 7 arerotated respectively, the cultivation vessel 2 is rotated around the 2axes. When the cultivation vessel 2 is rotated around the 2 axes, thedirections of the gravity applied to the cultivation cell lumpencapsulated in the cultivation vessel 2 are distributed. Thus, eachcell of the cell lump can multiply to all the directions. Also, the3-dimensional cultivation of the cultivation cell lump becomes possible.Also, the shearing force due to the liquid flow caused through therotation of the cultivation vessel 2 around the n axes is small. Theseparation of the cultivation cell lump and the isolation of each cellcan be prevented. Therefore, by cultivating the cell lump in thecultivation vessel 2 in the rotation state around the 2 axes, thecultivating of the tissue with the 3-dimensional structure is realized.

At this time, it is desirable that a differentiation factor like activinis added to the culture fluid when a desired tissue should beappropriately cultivated. The addition of the differentiation factor tothe culture fluid makes it possible to promote the differentiation ofthe cultivation cell and to cultivate a tissue appropriately.

Also, it is desirable a viscosity increasing material likemethylcellulose and collagen gel is added to the culture fluid. It isdesirable that the coefficient of viscosity of the culture fluid isadjusted such that the cultivation cell does not touch or contact thewall of the cultivation vessel 2 when the cultivation vessel 2 isrotated around the 2 axes. Thus, the sinking of the cultivation celllump is prevented.

FIG. 2 and FIG. 3 are photographs showing the outer appearance and crosssection of the cell lump cultivated by the method of cultivating a cellin the first embodiment. The cell lump is cultivated by the followingmethod. First, the A6 cell of a normal kidney of a male living body ofXenopus is adhesively cultivated for 2 weeks under the environment of23° C. in a cultivation flask. Subsequently, a part of the cultivationcell lump multiplied in a single layer state is peeled off from thecultivation surface of the adhesion flask and is moved to theabove-mentioned cultivation vessel 2. The cultivation vessel 2 is filledwith culture fluid. Subsequently, the cultivation vessel 2 is rotated bythe 3-dimensional klinostat 1 and the cultivation cell lump iscultivated in the cultivation vessel 2 in the floating state. Thecultivation is carried out for 2 weeks under the environment of 23° C.As shown in FIG. 2, spherical cell lump was observed. Also, the formedcell lump is sliced and the internal state of the cell lump is observed.At this time, as shown in FIG. 3, the tissue like the nephridium wasobserved. This experiment fact shows that the tissue with the3-dimensional structure can be formed by cultivating the cell lump bythe method of cultivating a cell in the first embodiment.

It is possible to apply the method of cultivating a cell in the firstembodiment to the manufacturing of an artificial organ. In this case, acultivation cell lump, which can be differentiated to a desired organ,is encapsulated in the cultivation vessel 2. The encapsulated cell lumpis cultivated while being rotated around the 2 axes to form theartificial organ with the 3-dimensional structure.

It should be noted that in the first embodiment, the cultivation vessel2 is rotated around the 2 axes but the cultivation vessel 2 may berotated around three or more rotation axes. In this case, like the abovecase of the 2-axis rotation, the directions of the gravity applied tothe cultivation cell lump through the rotation of the cultivation vessel2 are distributed, so that each cell can multiply to all the directions.Thus, the 3-dimensional cultivation of the cultivation cell lump becomespossible. Also, the shearing force of the liquid flow caused through therotation around the n axes in the cultivation vessel 2 becomes small, sothat the separation of the cultivation cell lump and the isolation ofeach cell can be prevented. By cultivating the cell lump in thecultivation vessel 2 in the rotation state of the cultivation vessel 2around two or more rotation axes, the cultivation of the tissue with the3-dimensional structure is realized.

(Second Embodiment)

In the method of cultivating a cell according to the second embodimentof the present invention, it is possible to cultivate a cell lump in thecultivation vessel by rotating the cultivation vessel filled with theculture fluid around the 2 axes, like the first embodiment. In thesecond embodiment, the cultivation is carried out in a state in whichfresh culture fluid is continuously supplied to the cultivation vesselrotated around the 2 axes so that it is possible to cultivate for a longterm.

FIG. 4 shows a cell cultivation apparatus 50 used in the method ofcultivating a cell according to the second embodiment of the presentinvention. The cell cultivation apparatus 50 is comprised of a3-dimensional klinostat 11, a cultivation vessel 12, a culture fluidtank 13 and a pump 14. The 3-dimensional klinostat 11 rotates thecultivation vessels 12 around 2 axes. The culture fluid and a cultivatedcell lump are encapsulated in the cultivation vessel 12. The cultivatedcell lump is not fixed to the cultivation vessel 12 but floats in theculture fluid. A differentiation factor like activin is added to theculture fluid to promote the differentiation to a desired tissue.

The culture fluid tank 13 accumulates the culture fluid which should besupplied to the cultivation vessel 12. The pump 14 pressurizes theculture fluid accommodated in the culture fluid tank 13 and supplies tothe cultivation vessel 12. The culture fluid supplied to the cultivationvessel 12 is returned to the culture fluid tank 13. Thus, the culturefluid for cultivating a cell lump is circulated.

It is desirable that the culture fluid tank 13 has a gas concentrationadjusting function to control the concentration of gas contained in theculture fluid. The optimization of the concentrations of oxygen andcarbon dioxide contained in the culture fluid is important in case ofthe cultivation of the cell lump. By adjusting the concentration of thegases contained in the culture fluid, especially, oxygen and the carbondioxide, the cell lump can be cultivated in a more desirableenvironment. More specifically, the culture fluid tank 13 is formed ofinterchangeable material of gas, and an atmosphere around the culturefluid tank 13 is adjusted. In this way, the gases contained in theculture fluid can be controlled to the desired concentrations.

The 3-dimensional klinostat 11 has the structure different from the3-dimensional klinostat 1 in the first embodiment, to supply the culturefluid to the cultivation vessel 12. The 3-dimensional klinostat 11 iscomprised of a main unit 15. A support 16 a and a support 16 b arejoined to the main unit 15. The support 16 a supports the outer frame 17rotatably. A rotary joint 18 and a motor 19 are joined to the support 16b. The rotary joint 18 supports the outer frame 17 rotatably. The motor19 drives the outer frame 17 through a power transfer mechanism (notshown) such as gears and a belt, to rotate the outer frame 17 around arotation axis 18 a. A rotary joint 20 and a motor 21 are joined to theouter frame 17. The rotary joint 20 supports the inner frame 22rotatably. The motor 21 drives the inner frame 22 through a powertransfer mechanism (not shown) such as gears and a belt to rotate theinner frame 22 around a rotation axis 20 a.

The above-mentioned cultivation vessel 12 is connected with the innerframe 22. The cultivation vessel 12 is in the neighborhood of anintersection of the rotation axis 18 a and the rotation axis 20 a. Thecultivation vessel 12 is rotated together with the inner frame 22. Whenthe outer frame 17 and the inner frame 22 are rotated respectively, thecultivation vessel 12 is rotated around 2 axes.

The supply of the culture fluid to the cultivation vessel 12 is carriedout through the rotary joint 18 and the rotary joint 20 from the pump14. The fresh culture fluid sent from the pump 14 reaches the rotaryjoint 18 through a supply pipe 23. The rotary joint 18 introduces theculture fluid into a supply pipe 24 which is rotated together with theouter frame 17. Also, the rotary joint 20 introduces the culture fluidinto a supply pipe 25 which is rotated together with the inner frame 22.The supply pipe 25 is connected with the cultivation vessel 12, and theculture fluid is supplied to the cultivation vessel 12 from the supplypipe 25.

In the same way, the discharge of the culture fluid to the culture fluidtank 13 is carried out through the rotary joint 18 and the rotary joint20 from the cultivation vessel 12. The cultivation vessel 12 dischargesthe culture fluid to a discharge pipe 26. Then, the culture fluiddischarged is introduced into a discharge pipe 27, which is rotatedtogether with the outer frame 17, through the rotary joint 20. Then, theculture fluid is introduced into a discharge pipe 28 which is put on astationary system through the rotary joint 18. The culture fluid isreturned to the culture fluid tank 13 through the discharge pipe 28.

FIG. 5 shows the rotary joint 18 in detail. The rotary joint 18 containsa fixed section 29 and a rotation section 30. The fixed section 29 andthe rotation section 30 are column bodies with different diameter. Therotation section 30 is inserted in the fixed section 29 rotatably. Thefixed section 29 is fixed to the support 16 b and the rotation section30 is fixed to the outer frame 17.

In the space between the fixed section 29 and the rotation section 30, afirst liquid introduction room 33 and a second liquid introduction room34 are formed by seals 31 and 32. The first liquid introduction room 33and the second liquid introduction room 34 are separated by the seal 31.The second liquid introduction room 34 is sealed from the external bythe seal 32. The supply pipe 24 is connected with the first liquidintroduction room 33 and the discharge pipe 27 is connected with thesecond liquid introduction room 34.

A first hole 35 opened for the first liquid introduction room 33 and asecond hole 36 opened for the second liquid introduction room 34 areprovided for the rotation section 30. The first hole 35 is connectedwith the supply pipe 24 and the second hole 36 is connected with thedischarge pipe 27.

The rotary joint 18 having the above structure connects the supply pipe23 and the supply pipe 24 and connects the discharge pipe 27 and thedischarge pipe 28, regardless of the angle between the support 16 b andthe outer frame 17.

The rotary joint 20 has the same structure as the rotary joint 18, andconnects the supply pipe 24 and the supply pipe 25 and connects thedischarge pipe 26 and the discharge pipe 27, regardless of the anglebetween the outer frame 17 and the inner frame 22.

In this way, the culture fluid is supplied to the cultivation vessel 12through the rotary joint 18 from outside the 3-dimensional klinostat 11through the rotary joint 20, and the culture fluid is discharged fromthe cultivation vessel 12 into outside the 3-dimensional klinostat 11.

In case of supplying the culture fluid to the cultivation vessel 12 fromthe external, when the culture fluid flows, the disorder of the liquidflow can be caused in the cultivation vessel 12. The disorder of theliquid flow introduces the separation of the cell lump to prevent thecultivation of the 3-dimensional tissue. For this reason, thecultivation vessel 12 has the structure to restrain an influence of thedisorder of the liquid flow.

FIG. 6 shows the cultivation vessel 12. The inside of the cultivationvessel 12 is separated into a flow path room 38 and a cultivation room39 with a separation member 37. The flow path room 38 is connected withthe supply pipe 25 to supply the culture fluid and the discharge pipe 26to discharge the culture fluid. A lot of holes (not shown) are providedfor the separation member 37, so that the exchange of the culture fluidcan be carried out between the flow path room 38 and the cultivationroom 39 through the holes. The cell lump 40 is put in the floating statein the cultivation room 39 and the cultivation of the cell lump 40 iscarried out in the cultivation room 39.

In the cultivation vessel 12 with the above structure, the culture fluiddoes not flow directly into the cultivation room 39 where thecultivation of the cell lump 40 is carried out. In this way, theinfluence of the disorder of the liquid flow by the culture fluidflowing into the cultivation vessel 12 on the cultivation of the celllump 40 can be restrained.

The cell cultivation apparatus according to the second embodiment of thepresent invention, can cultivate a tissue with a 3-dimensionalstructure, like the first embodiment. Also, in the second embodiment, itis possible to cultivate a cell for long term.

It should be noted that the culture fluid used for the cultivation inthe second embodiment is circulated. However, the culture fluiddischarged from the cultivation vessel 12 may be dumped just as it is.In this case, the discharge pipe 28 used to discharge the culture fluidfrom the cultivation vessel 12 is not connected with the culture fluidtank 13.

(Third Embodiment)

The method of cultivating a cell according to the third embodiment ofthe present invention is especially applied to manufacture an artificialorgan.

The method of cultivating a cell according to the third embodiment ofthe present invention is similar to the first and second embodiments inthat the cultivation vessel filled with the culture fluid is rotatedaround 2 axes, and the cell is cultivated in the cultivation vessel.

The third embodiment is different from the first and second embodimentsin that a cell lump is adhered to the artificial matrix which is fixedon the cultivation vessel and is cultivated in that state. In the thirdembodiment, gravity is applied 3-dimensionally and the 3-dimensionalcultivation becomes possible in which the cell lump is adhered uniformlyto the artificial matrix with the 3-dimensional structure. Also, theshearing force by the fluid stirring caused by the 2 axes rotation ofthe cultivation vessels is small, so that the separation and damage ofthe cell lump adhered and densified can be prevented.

The cultivation of the cell lump on the artificial matrix makes itpossible to cultivate a large-sized artificial organ. In order tocultivate a large-sized artificial organ, it is necessary to cultivate alarge-sized cell lump. However, it is difficult to keep a large-sizedcell lump in the perfect floating state, and it is easy for damage to beadded to the cell lump when the cell lump is large-sized. By cultivatingthe cell lump on the artificial matrix which is fixed on the cultivationvessel, the damage of the cell lump can be prevented and the cultivationof the large-sized cell lump becomes possible. At this time, theartificial matrix supports the cell lump fixedly and functions as askeleton in the cultivation of the cell lump to form a part of theartificial organ.

In the third embodiment, the cultivation vessel 41 shown in FIG. 7 isused instead of the cultivation vessel 12 used in the second embodiment.The cultivation vessel 41 is separated into the flow path room 43 andthe cultivation room 44 by the separation member 42. The flow path room43 is connected with the supply pipe 25 to supply the culture fluid andthe discharge pipe 26 to discharge the culture fluid. A lot of holes(not shown) are provided for the separation member 42, and the exchangeof the culture fluid is carried out between the flow path room 43 andthe cultivation room 44 through the holes.

The artificial matrix 45 and the artificial matrix support sections 46and 47 are stored in the cultivation room 44. For example, theartificial matrix 45 is formed of sponge collagen. The artificial matrix45 is used for a skeleton when a cell lump (not shown) is cultivated,and a cell lump is cultivated in the state adhered to the artificialmatrix 45. The artificial matrix support sections 46 and 47 hold orsupport the artificial matrix 45 fixedly to the wall of the cultivationvessel 41. The cell lump cultivated by using the artificial matrix 45and the artificial matrix support sections 46 and 47 is fixedlysupported in the culture fluid.

A modification of the cell cultivation apparatus according to the thirdembodiment of the present invention has the same as the secondembodiment and the detailed description is not carried out.

In the third embodiment, the cultivation of a tissue is carried out asfollows. The cultivation cell is adhered to the artificial matrix 45.The artificial matrix 45 is fixed on the wall of the cultivation room 44by the artificial matrix support sections 46 and 47. The inside of thecultivation vessel 41 is filled with the culture fluid. The cultivationvessel 41 is rotated around the 2 axes by the 3-dimensional klinostat11, and the cultivation is started. During the cultivation, the pump 14supplies the culture fluid to the cultivation vessel 41 from the culturefluid tank 13. The culture fluid discharged from the cultivation vessel41 is returned to the culture fluid tank 13. The culture fluid flowsthrough the cultivation room 44 3-dimensionally and the gravity isapplied to the artificial matrix 45 from all directions. Thus, the cellmultiplies 3-dimensionally on the surface with the artificial matrix 45.

The tissue composed of the artificial matrix 45 and the cultivation celllump is taken out from the cultivation vessel 41 as an artificial organ.It is possible to replay or recover the function of an internal organ bytransplanting the artificial organ into a body or installing it out ofthe body.

It is desirable that the artificial matrix pipe 48 is provided for theartificial matrix 45 as shown in FIG. 8, to cultivate a large-sizedartificial organ. The one end of the artificial matrix pipe 48 isconnected with the supply pipe 25 shown in FIG. 4, and the other end isconnected with the discharge pipe 26. Thus, the culture fluid is passedinside the artificial matrix pipe 48. When the tissue to be formed onthe artificial matrix 45 is large-sized, it is difficult to supply thematerial necessary for multiplication into the tissue. The artificialmatrix pipe 48 in which the culture fluid is passed acts as anartificial vein and the material necessary for multiplication issupplied inside the tissue. When the artificial matrix pipe 48 isprovided, the artificial matrix pipe 48 can support the artificialmatrix 45. As shown in FIG. 8, the artificial matrix support sections 46and 47 do not have to be always provided.

The third embodiment of the cell cultivation apparatus according to thepresent invention can be form the tissue with the 3-dimensionalstructure, like the first and second embodiments. Also, in the thirdembodiment, it is possible to cultivate a large-sized tissue.

(Fourth Embodiment)

The 3-dimensional klinostat according to the fourth embodiment of thepresent invention is used as a part of a plant growing apparatus. Theplant growing apparatus in the fourth embodiment rotates a growth vesselaccommodating a plant around the 2 axes and the gravity applied to theplant as the growth object is distributed into 3-dimensional directions.The plant growing apparatus in the fourth embodiment is used to confirmthe physiological activity of the plant under the environment thegravity is distributed 3-dimensionally.

The plant growing apparatus in the fourth embodiment which rotates thegrowth vessels around the 2 axes has the same structure as the cell lumpcultivation apparatus in the first embodiment approximately but isdifferent from the first embodiment on the following points.

The growth vessel 51 shown in FIG. 9 is first used instead of thecultivation vessel 2 used in the first embodiment. The growth plant isaccommodated in the growth vessel 51. The details of the growth vessel51 are later mentioned.

Second, not the culture fluid but growth liquid is accumulated in thetank 3 shown in FIG. 4. The growth liquid is controlled to contain thematerial necessary to grow a plant. The growth liquid accumulated in thetank 3 is supplied to the growth vessel 41 through the rotary joint 8and the rotary joint 12. The excessive growth liquid is discharged fromthe growth vessel 41 and is discharged to the tank 3 through the rotaryjoint 8 and the rotary joint 12. It is desirable that the tank 3 has thefunction to adjust the concentration of the material necessary to growthe plant the oxygen concentration and pH of growth liquid.

As shown in FIG. 9, the above-mentioned growth vessel 51 is connectedwith a supply pipe 18 and a discharge pipe 19. The growth liquid issupplied from the supply pipe 18 to the growth vessel 51 and theexcessive growth liquid is discharged from the discharge pipe 19. Thegrowth vessel 51 has a water keeping member 52 like lock wool therein.The growth liquid supplied to the growth vessel 51 is filtered into thewater keeping member 52. The plant 53 as the growing object is plantedin the water keeping member 52, and the plant 53 absorbs the materialnecessary to grow from the growth liquid filtered into the keepingmember 52.

The plant growing apparatus in the fourth embodiment supplies the growthliquid to the growth vessel 51 in the state which the growth vessels 51is rotated around the 2 axes, and discharges the growth liquid from thegrowth vessel 51, so that the growth liquid in the growth vessel 51 canbe exchanged. The plant growing apparatus is not needed to stop therotation of the growth vessel 51 to supply, discharge or exchange thegrowth liquid to the growth vessel 51 and is suitable to grow a plantunder the rotation environment for a long term.

It should be noted that in the fourth embodiment, the growth vessel 51is rotated around the 2 axes. However, the growth vessel 51 may berotated around two or more axes.

Also, the plant growing apparatus in the fourth embodiment can beapplied to the growth of a microorganism like mold. In this case, themicroorganism is stored in the growth vessel 51 instead of plant 53 andis grown.

(Fifth Embodiment)

The 3-dimensional klinostat according to the fifth embodiment of thepresent invention is used as a part of an aquatic organism growingapparatus. The aquatic organism growing apparatus in the fifthembodiment rotates the growth vessel accommodating the aquatic organismas the growth object around the 2 axes. The gravity applied to theaquatic organism as the growth object is distributes in all thedirections 3-dimensionally. The aquatic organism growing apparatus inthe fifth embodiment is used to confirm the activated course of thephysiological activity of the aquatic organism under the environment inwhich the gravity is distributes in all the directions 3-dimensionally.

The aquatic organism growing apparatus in the fifth embodiment hassubstantially the same structure as the cell lump cultivation apparatusin the first embodiment and differs from the first embodiment in thefollowing points.

First, the growth vessel 54 shown in FIG. 10 is used instead of thecultivation vessel 2 in the first embodiment. The aquatic organism isaccommodated in the growth vessel 54. It is desirable that the aquaticorganism to be grown may be any of an animal, a plant and amicroorganism, e.g., a newt, a fish, a aquatic plant. The details of thegrowth vessel 54 are mentioned later.

Second, breeding water is accumulated in the tank 3 shown in FIG. 4instead of the culture fluid. The breeding water is controlled tocontain the material necessary to grow the aquatic organism. When theaquatic organism is an animal, the bait of the aquatic organism is mixedwith the breeding water and is supplied to the growth vessel 54. Thebreeding water is accommodated in the tank 3 and is supplied to thegrowth vessel 54 through the rotary joint 8 and the rotary joint 12. Theexcessive breeding water is discharged from the growth vessel 54 and isdischarged to the tank 3 through the rotary joint 8 and the rotary joint12. It is desirable that the tank 3 has a gas exchange function toadjust the gas concentration of the breeding water, a control functionof pH, and a removal function to remove egesta from the aquaticorganism.

As shown in FIG. 10, the above-mentioned growth vessel 54 contains aflow path room 55 and a growth room 56. The supply pipe 18 whichsupplies the breeding water and the discharge pipe 19 which dischargesthe breeding water are connected with the flow path room 55. The flowpath room 55 is separated from the growth room 56 by a separation member57. A lot of holes are provided for the separation member 57. Throughthe holes, the exchange of the culture fluid between the flow path room55 and the growth room 56 is carried out. The aquatic organism 58 is putin the growth room 56 and the cultivation for the growth of the aquaticorganism 58 is carried out in the growth room 56.

When eggs of the aquatic organism 58 are grown, an egg holder 59 ispreferably stored in the growth room 56, as shown in FIG. 11. Aplurality of egg storage rooms 60 which are cylindrical holes areprovided for the egg holder 59, and the eggs 61 are stored in the eggstorage rooms 60 and are fixed therein. Neighbor egg storage rooms 60are connected with each other by flow paths 62, and the flow path 63 isprovided for ones of the egg storage room 60 which face to the flow pathroom 55 and connected with the flow path room 55. The breeding water issupplied to each egg storage room 60 through the flow path 62 and theflow path 63.

The aquatic organism growing apparatus in the fifth embodiment suppliesthe breeding water to the growth vessel 54 and discharges from thegrowth vessel 54, in the rotation state of the growth vessels 54 aroundof the 2 axes, so that the breeding water in the growth vessel 54 can beexchanged. The aquatic organism growing apparatus needs not to stop therotation of the growth vessel 54 to supply the breeding water to thegrowth vessel 54. Therefore, it is suitable to grow the aquatic organismfor a long term under the rotation environment.

It should be noted that in the fifth embodiment, the growth vessels 54are rotated around the 2 axes. However, the growth vessel 54 can berotated around 2 or more axes.

(Sixth Embodiment)

The 3-dimensional klinostat according to the sixth embodiment of thepresent invention is used as a part of the animal growing apparatus. Theanimal growing apparatus in the sixth embodiment rotates the growthvessel, which accommodates an animal as an growth object, around the 2axes to distribute the direction of the gravity applied to the animal asthe growth object 3-dimensionally. The animal growing apparatus in thesixth embodiment is used to confirm the activated course of thephysiological activity of the animal under the environment in which thedirection the gravity is distributed 3-dimensionally.

The animal growing apparatus in the sixth embodiment has substantiallythe same structure as the cell lump cultivation apparatus in the firstembodiment but differs from the first embodiment in the followingpoints.

First, the growth vessel 71 shown in FIG. 12 is used instead of thecultivation vessel 2 used in the first embodiment. An animal to begrown, e.g., a mouse is housed in the growth vessel 71. The details ofthe growth vessel 71 are later mentioned.

Second, it is not the culture fluid but drinking water that isaccumulated in the tank 3 shown in FIG. 4. The drinking wateraccumulated in the tank 3 is supplied to the growth vessel 71 throughthe rotary joint 8 and the rotary joint 12.

Third, a discharge pipe 21 is not connected with the tank 3. The olddrinking water after being supplied to the growth vessel 71 isdischarged to the discharge pipe 21 through the rotary joint 12 and therotary joint 8. The drinking water is discharged to the discharge pipe21 and is dumped away just as it is.

As shown in FIG. 12, a growth room 72 and an excrement processing room73 are provided for the above-mentioned growth vessel 71. The growthroom 72 accommodates the animal 75 as a growth object. It is desirablethat the growth room 72 has such a size as the animal 75 cannot changethe direction of itself. A bait box 76 and a drinking water supplyvessel 77 are provided for the growth room 72. The bait box 76 suppliesthe bait to the animal 75. The drinking water supply vessel 77 isconnected with the supply pipe 18. As described above, the supply pipe18 is connected with the tank 3 through the rotary joint 8 and therotary joint 12. The supply pipe 18 supplies the drinking wateraccumulated in the tank 3 to the drinking water supply vessel 77. Thedrinking water supply vessel 77 supplies the drinking water suppliedfrom the supply pipe 18 to the animal 75. Also, the drinking watersupply vessel 77 is connected with the discharge pipe 19. The olddrinking water after being supplied to the drinking water supply vessel77 is discharged from the discharge pipe 19 through the rotary joint 12and the rotary joint 8 to the discharge pipe 21. The drinking water isdischarged to the discharge pipe 21 and is thrown or dumped away just asit is. The growth room 72 is separated from the excrement processingroom 73 by a network 74.

An adhesive sheet 78 and a moisture absorbing member 79 are provided forthe excrement processing room 73. The adhesive sheet 78 captures theshit discharged from the animal 75. The moisture absorbing member 79absorbs urine discharged from the animal 75. The sanitary state of thegrowth room 72 is maintained by the adhesive sheet 78 and the moistureabsorbing member 79.

The animal growing apparatus described above in the sixth embodimentsupplies drinking water to the growth vessel 71 in the rotation state ofthe growth vessels 71 around the 2 axes, and discharges the drinkingwater from the growth vessel 71, so that the drinking water supplied tothe growth vessel 71 can be exchanged. The animal growing apparatusneeds not to stop the rotation of the growth vessel 71 to supplydrinking water to the growth vessel 71 and to discharge or to exchange.It is suitable to grow the animal for a long term under the rotationenvironment.

It should be noted that in the sixth embodiment, the growth vessels 71are rotated around the 2 axes but the growth vessel 71 can be rotatedaround two or more axes.

The practical technique to cultivate the tissue with the 3-dimensionalstructure through the cultivation of the cell is provided according tothe present invention.

Also, according to the present invention, a technique that theseparation of the cell lump can be prevented is provided when the tissuewith the 3-dimensional structure is formed.

Also, according to the present invention, a technique that can cultivatethe cell for a long term is provided when the tissue with the3-dimensional structure is formed.

Also, according to the present invention, a technique that theenvironment of the cultivation of the cell can be optimized is providedwhen the tissue with the 3-dimensional structure is formed.

Also, according to the present invention, a technique which cancultivate a large-sized tissue with a 3-dimensional structure isprovided.

What is claimed is:
 1. A method of growing and cultivating cells,comprising: a) encapsulating the cells and a fluid in a vessel, whereinsaid vessel is part of a rotating unit; b) rotating said vessel arounddifferent n axes, wherein n is an integer greater than 1; and c)continuously flowing fluid through a flow path formed between the vesseland an external fluid tank, while said vessel is rotating.
 2. The methodaccording to claim 1, further comprising adjusting a coefficient ofviscosity of said fluid such that said cells do not touch and attach tosaid vessel.
 3. The method of claim 1, wherein said c) continuouslyflowing fluid comprises: pumping fluid into said vessel; circulatingsaid fluid within said vessel; and withdrawing fluid from said vessel.4. A method of growing and cultivating cells, comprising: a)encapsulating the cells and a fluid in a vessel, wherein said vessel ispart of a rotating unit, said rotating unit comprising an inner frame,an outer frame and a base, said inner frame connected to said outerframe via a first rotary joint, and said outer frame connected to saidbase via a second rotary joint, wherein said vessel is connected to saidinner frame; b) rotating said vessel around different n axes, wherein nis an integer greater than 1, wherein said rotating comprises rotatingthe inner frame about a first one of the different n axes via said firstrotary joint, and rotating the outer frame about a second one of thedifferent n axes via said second rotary joint; and c) continuouslyflowing the fluid through a flow path defined between the vessel and anexternal fluid tank, while said vessel is rotating, wherein said flowpath goes through the first and second rotary joints within the rotatingunit.